Metronidazole is commonly used for the treatment of giardiasis, an intestinal infection caused by the protozoan parasite Giardia lamblia. Reports of clinical resistance to this drug and recurrent infections underscore the need for alternative therapeutics. Since G. lamblia trophozoite proliferation in the intestine is intimately associated with the symptoms of giardiasis, we developed a miniaturized proliferation assay amenable to high-throughput screening for compounds which inhibit trophozoite proliferation. A pilot screen of ~1500 bioactive compounds uncovered 50 small molecules which significantly depressed or arrested growth. Many of these compounds were not known to inhibit G. lamblia. We propose to use this simple, high-throughput assay to screen a large collection of compounds. This screen will be conducted simultaneously with two G. lamblia assemblages (genotypes) to identify compounds which inhibit both assemblages, and compounds which differentially perturb proliferation of assemblage A or B. Inhibitors confirmed in a secondary screen will be categorized with respect to their molecular target, thus identifying new druggable pathways in G. lamblia. The dual screening will enable the identification of compounds which differentially affect G. lamblia assemblages infecting humans. Based on the mode of action of such compounds, we will be able to formulate hypotheses on the molecular basis of phenotypic differences between assemblage. This proposal is submitted in response to the R21 Developmental/Exploratory grant solicitation which is intended to support projects which are in their early phase and are based on novel experimental systems. Specifically, we will use a trophozoite proliferation assay to screen, in 384-well plates, small molecule libraries with an assemblage A and an assemblage B isolate of G. lamblia. Inhibitors identified in the high-throughput screen will be verified with additional assemblage A and assemblage B isolates. Microscopy and flow cytometry will be used to investigate the target of selected, confirmed inhibitors. PUBLIC HEALTH RELEVANCE: Giardia lamblia is a common cause of intestinal infections. We propose to screen large collections of small molecules to identify new leads for future development of drugs against this parasite.